Malaria is a mosquito-borne disease and, despite many years of research, remains a major global health problem causing illness and death that disproportionately affects developing countries. The worldwide incidence of malaria is estimated by the World Health Organization to be approximately 300 to 500 million clinical cases annually, with at least one million deaths. The majority of these are young children [WHO Malaria report 2008, Snow et al. 2005, Guerra et al. 2008 and Hay Si et al. 2009]. The emergence of insecticide-resistant mosquito vectors and multi-drug resistant parasites has contributed to resurgences of the disease.
A number of anti-malarial vaccines has been used to combat the disease with some ameliorative effects [Bejon P. et al., 2009] and new vaccines and treatments regularly appear on the market. The determination of efficacy from these is difficult and compounded by the generally poor health status of many of the trial groups. To date, none has appeared to be highly protective but any vaccine that reduces morbidity and mortality is a valuable new tool in the fight against malaria. The most severe form of the disease is caused by Plasmodium falciparum [Casteels, P. C. et al. 1989, Chopra, L. 1993]. Infection begins when malarial sporozoites are injected by mosquitoes into the host and within minutes parasites invade hepatocytes, where they multiply and differentiate into the next stage of the life-cycle, the merozoites. The merozoites emerging from the hepatocytes invade red blood cells leading to clinical illness [Sturm A. et al. 2006]. The most advanced vaccine candidate, designated RTS, S/AS02A, [Bejon P. et al. 2009] is based on the major sporozoite surface antigen. However, this candidate vaccine, currently in Phase 3 clinical trials, has shown only 30-65% efficacy in field studies [Dauville D. et al. 2010] and a vaccine with higher levels of protection is still sought. Over a period of time, people living in areas where malaria is endemic develop immunity to clinical disease caused by P. falciparum and immunoglobulin G taken from immune adults has been shown to reduce parasite density and clinical symptoms when administered to children with clinical malaria [Cohen et al. 1961, Bauharoun-Tayoun H. et al. 1990, Sabchareon A. et al. 1991]. Thus, proteins expressed during the blood-stage of the life cycle are good candidates for inclusion in a vaccine [Good M. F. 2001. Malkin E. at al. 2007], as a blood-stage vaccine would reduce or prevent severe illness and complications of the disease.
Many microbial pathogens, viral or bacterial, include a secondary host in their life-cycle which in many cases shows no overt signs of disease in contrast to that of the host with which the disease is primarily associated. The secondary host in these cases provides an environment which is physiologically permissive for growth and differentiation of the pathogen, frequently without any detrimental effect to itself. In the mosquito, malarial association however, where the mosquito acts as an efficient vector for transmission of the Plasmodium parasite to mammals, the mosquito nevertheless presents multiple barriers to the unrestricted growth of the parasite minimising multiplication in this part of the life-cycle [Warburg and Miller, 1991; Beier, 1998]. These barriers arise from a number of factors, including anatomical features of the mosquito host and physiological incompatibilities between insect and parasite.
It is probable that the innate immune system of the mosquito plays a significant part in this restriction of parasite growth or development and may be the predominant source of this controlling effect. Insects in general respond to bacterial or fungal infections by rapidly synthesizing a battery of potent antimicrobial peptide factors [Hetru et al., 1998]. The cloning of genes encoding these peptides in model insect species, particularly the fruit fly Drosophila melanogaster, has provided powerful tools with which to explore the mechanisms involved in the elicitation of the insect innate immune response [Hoffmann et al. 1996]. Recently, progress has been made in applying this basic knowledge of invertebrate immunity to dipteran insects of medical importance [Richman & Kafatos, 1996]. In particular, interest has focused on the mosquito Anopheles gambiae which is the most important african vector of the human malaria parasite Plasmodium falciparum and on Aedes aegypti the transmitter of yellow fever. This species is also a vector of a number of other protozoan and metazoan parasites. Initial studies of humoral immunity in both of these insect species has led to the purification of a group of antimicrobial peptides known as “defensins” and to the cloning of defensin-encoding cDNAs [Chalk et al. 1994; Lowenberger et al. 1995; Cho et al. 1996; Richman and Kafatos, 1996]. Both A. gambiae and A. aegypti respond to bacterial infections through the rapid induction of defensin RNA and protein [Lowenberger et al. 1995; Richman et al. 1996].
Further studies in A. gambiae have shown that humoral immune mechanisms are activated in multiple host mosquito tissues and at multiple time points during the course of infection of the mosquito by the rodent malarial parasite, Plasmodium berghei [Richman et al. 1997; Dimopoulos et al. 1998]. The fact that immune-competent mosquitoes nevertheless provide a physiological milieu at least partially permissive for the growth and differentiation of Plasmodium, represents an intriguing biological phenomenon of great significance for human health.
The extent to which endogenous humoral effector molecules may act to limit parasite development or growth in insects is largely unknown. Defensins have been shown to have effects on certain stages of Plasmodium either in vitro or when injected into the haemolymph of infected mosquitoes [Shahabuddin et al. 1998]. However, a microorganism or parasite invading a dipteran insect will probably encounter multiple humoral defense factors which may act synergistically.
Calvo et al. (2009) isolated an anti-microbial peptide, a cecropin homologue, from the salivary gland of the mosquito A. darlingi. Cecropins are powerful antimicrobial compounds present in insect haemolymph and are also found in pig intestines [Boman, H. G. 1991; Boman, H. G., et al. 1991; Lee, J. Y., et al. 1989]. They are strongly cationic, amphipathic, α-helical peptides with 35 to 39 residues and are notably active not only against certain gram-positive bacteria but also against gram-negative bacteria. Many other cationic peptides, such as defensins from leukocyte granules and magainins from frog skin, have also evoked considerable interest in recent years [Casteels, P. C. et al. 1989; Diamond, G., et al. 1991; Lehrer, R I., et al. 1991; Nakamura, T. et al. 1988; Parra-Lopez, C., 1993; Zaslof, M. 1987; Zasloff, M., B. et al. 1988]. However, cecropins are 10 to 30 times more active than defensins and magainins, against Escherichia coli and Pseudomonas aeruginosa [Boman, H. G. 1991; Wade, D. A. et al. 1990].
The strongly cationic N-terminal, amphipathic helix of cecropins has been shown to be necessary for effective binding to bacterial membranes allowing them to cause instantaneous lysis of bacterial cells, through disintegration of the cytoplasmic membrane [Christensen, B. J. 1988]. Cecropins form ion channels in artificial membranes [Christensen, B. J. et al. 1988], and cecropin dimers can be predicted by computer modeling to form channel-containing regular lattice structures on the membrane surface [Durell, S. R., et al. 1992]. However, such channels probably develop only when the cecropin density is high and disintegration of the membrane takes place [Christensen, B. J. et al. 1988; Durell, S. R., et al. 1992]. Accordingly, the lethal target of cecropins in these studies was the bacterial cytoplasmic membrane.
Cecropin molecules possess amphipathicity which allows them to interact simultaneously with lipid-like and negatively charged molecules through their cationic region, so as to attach themselves to the microbial membrane (Ganz T, and Lehrer R I, 1998). The initial contact between the peptide and the target organism is electrostatic. Their amino acid composition, amphipathicity, cationic charge and size allow them to insert into the membrane bilayer to form pores by attaching themselves as a ‘carpet’ to penetrate the membrane (Giuliani et al., 2007).
With the exception of Bombyx and Aedes cecropins, all other insect cecropins so far characterized are C-terminally amidated. This post-translational modification has been considered necessary for the full anti-microbial activity of the molecule (Li et al. 1988; Hara et al. 1994), and may protect the peptide from carboxypeptidase digestion (Callaway et al. 1993). The presence of a glycine residue at the end of the deduced amino acid sequence of A. Gambiae cecropin suggested C-terminal amidation via terminal glycine removal (Bradbury & Smyth, 1991) produced a fully active peptide.